Bottom Line:
By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications.In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators.We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.

ABSTRACTXAF1 is a tumour suppressor gene that compromises cell viability by modulating different cellular events such as mitosis, cell cycle progression and apoptosis. In cancer, the XAF1 gene is commonly silenced by CpG-dinucleotide hypermethylation of its promoter. DNA demethylating agents induce transcriptional reactivation of XAF1, sensitizing cancer cells to therapy. The molecular mechanisms that mediate promoter CpG methylation have not been previously studied. Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner. By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications. In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators. We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.

f3: Features of the XAF1 promoter.(a) The XAF1 promoter visualized in the UCSC genome browser. The picture illustrates the CpG-methylation status from different types of cell lines. Additionally, the profiles of several histone posttranslational modifications such as H3K4Me1, H3K4Me3 and H3K27Ac are presented from different cell lines. Several transcription factor binding sites obtained from ChIP-Seq data are also shown. At a higher resolution, the CTCF binding site in the XAF1 promoter in glioblastoma and fibroblast cells is shown. (b) Schematic representation of the XAF1 promoter showing the CpG-dinucleotide positions from −22 to −500 bp relative to the transcription start site and the previously described binding sites for IRF-1, ISRE, p53 and the uncharacterized CTCF binding site. Histone posttranslational modification (HPM).

Mentions:
CTCF is known to regulate the expression of diverse tumour suppressor genes by directly binding to promoter sequences28. We searched for transcription binding sites in a window of −/+ 10 bp of the DNA sequence adjacent to each CpG that was demethylated as a consequence of epigenetic modifiers. Interestingly, we identify a putative CTCF binding site that overlapped the CpG dinucleotide located at −388 bp relative to the transcription start site (Fig. 2b). Supporting the relevance of this site, its presence was confirmed in an experimentally validated CTCF-binding site database33 (Fig. 3a,b). To experimentally validate this, ChIP assays were performed in MCF-7 cells after stimulation with TNF-α or IFN-α. As shown in Fig. 2c, in basal conditions, we could not find a detectable association of CTCF with the putative CTCF binding site in the XAF1 promoter. This result could be explained by a methylation-sensitive CTCF binding mechanism. To directly test this, we exposed the cells to demethylating agents before stimulation with TNF-α or IFN-α. As expected, the association of CTCF with the XAF1 promoter was detected only after DNA demethylation and stimulation with TNF-α or IFN-α (Fig. 2c). This observation correlated with an increased transcriptional activation when the cells were previously exposed to the epigenetic modifiers (Fig. 2c, third panel). Additionally, we validated this CTCF binding site using an additional cell line. As in MCF-7 cells, we observed a dramatic increase in the interaction of CTCF with the XAF1 promoter when the cells were stimulated with either TNF-α or IFN-α after exposure to demethylating agents (Supplementary Fig. 1c). These results support a methylation-sensitive association of CTCF with the XAF1 promoter.

f3: Features of the XAF1 promoter.(a) The XAF1 promoter visualized in the UCSC genome browser. The picture illustrates the CpG-methylation status from different types of cell lines. Additionally, the profiles of several histone posttranslational modifications such as H3K4Me1, H3K4Me3 and H3K27Ac are presented from different cell lines. Several transcription factor binding sites obtained from ChIP-Seq data are also shown. At a higher resolution, the CTCF binding site in the XAF1 promoter in glioblastoma and fibroblast cells is shown. (b) Schematic representation of the XAF1 promoter showing the CpG-dinucleotide positions from −22 to −500 bp relative to the transcription start site and the previously described binding sites for IRF-1, ISRE, p53 and the uncharacterized CTCF binding site. Histone posttranslational modification (HPM).

Mentions:
CTCF is known to regulate the expression of diverse tumour suppressor genes by directly binding to promoter sequences28. We searched for transcription binding sites in a window of −/+ 10 bp of the DNA sequence adjacent to each CpG that was demethylated as a consequence of epigenetic modifiers. Interestingly, we identify a putative CTCF binding site that overlapped the CpG dinucleotide located at −388 bp relative to the transcription start site (Fig. 2b). Supporting the relevance of this site, its presence was confirmed in an experimentally validated CTCF-binding site database33 (Fig. 3a,b). To experimentally validate this, ChIP assays were performed in MCF-7 cells after stimulation with TNF-α or IFN-α. As shown in Fig. 2c, in basal conditions, we could not find a detectable association of CTCF with the putative CTCF binding site in the XAF1 promoter. This result could be explained by a methylation-sensitive CTCF binding mechanism. To directly test this, we exposed the cells to demethylating agents before stimulation with TNF-α or IFN-α. As expected, the association of CTCF with the XAF1 promoter was detected only after DNA demethylation and stimulation with TNF-α or IFN-α (Fig. 2c). This observation correlated with an increased transcriptional activation when the cells were previously exposed to the epigenetic modifiers (Fig. 2c, third panel). Additionally, we validated this CTCF binding site using an additional cell line. As in MCF-7 cells, we observed a dramatic increase in the interaction of CTCF with the XAF1 promoter when the cells were stimulated with either TNF-α or IFN-α after exposure to demethylating agents (Supplementary Fig. 1c). These results support a methylation-sensitive association of CTCF with the XAF1 promoter.

Bottom Line:
By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications.In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators.We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.

ABSTRACTXAF1 is a tumour suppressor gene that compromises cell viability by modulating different cellular events such as mitosis, cell cycle progression and apoptosis. In cancer, the XAF1 gene is commonly silenced by CpG-dinucleotide hypermethylation of its promoter. DNA demethylating agents induce transcriptional reactivation of XAF1, sensitizing cancer cells to therapy. The molecular mechanisms that mediate promoter CpG methylation have not been previously studied. Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner. By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications. In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators. We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.